Our goals are to determine the structural basis for HIV-1 protease resistance; that is, how moiecular/structural changes in protease acquired during the development of drug resistance maintain protease function while facilitating inhibitor resistance. We have used TL-3, a protease inhibitor developed during the previous funding period, as a molecular probe to better understand structure and function relationships of HIV-1 protease as drug resistance evolved. These studies led to isolation and characterization of a panel of TL-3 resistant HIV variants that demonstrated varied degrees of resistance to TL-3 and to a number of other protease inhibitors. Our panel of protease mutants, and others that will be isolated, will be utilized to investigate molecular and structural changes in protease necessary for maintenance of protease function and development of inhibitor cross-resistance. The findings will aid our colleagues in this Program Project in compound development and validation of computational modeling of the development of drug resistance. To this end, we propose the following Specific Aims: 1. Perform tissue culture and enzymatic analyses to determine the relative susceptibility of wild-type and mutant HIV-1 to drug treatment as a function of specific structural changes in protease. 2. Use the panel of drug-resistant HIV-1 proteases in combination with phage display libraries to define the molecular basis for substrate specificity. 3. Use the panel of drug-resistant HIV-1 proteases in combination with RNA aptamer libraries to attempt to define novel structural elements of wild-type and drug-resistant proteases that can be targeted to modify/disrupt function. 4. Assess novel protease inhibitors developed by our collaborators.